Trypan blue: dye exclusion viability test

Cell viability is an index of the “health state” of the cells in a sample.

Before carrying out any cell experiments aimed at characterizing morphology, pathophysiological responses, and so on, you need to be certain that the cells in your culture are viable.

Furthermore, cell viability may be of interest after exposure of the cultured cells to an agent whose cytotoxic effect is to be determined (chemotherapy drugs) and/or excluded (cosmetics or food additives and so on).

Viability is often defined as the number, or rather the percentage, of healthy cells in a sample’s overall cell population.

Nowadays, several viability tests are used, but the widely used trypan blue remains one of the simplest and fastest to perform.

Trypan blue

Trypan Blue (Benzamine Blue, Naphthylamine Blue, Niagara Blue) was synthesized in the early twentieth century as a derivative of toluene.

Its name, Trypan, depends on the fact that it is toxic to the trypanosome parasite (analogues of trypan blue are currently used as pharmacological agents).

Trypan Blue is commonly used for the study of cell viability by a method referred to as exclusion staining.

In fact, membranes are normally impermeable to this dye in healthy cells, while dead cells, both apoptotic and necrotic or autophagocytotic, are colored blue (without the possibility of distinction). It is therefore a rather crude and unselective test, but still widely used as it is quick and easy.

Traditionally this test involves the cell count (with distinction between live and dead cells) with a hemacytometer viewed under an optical microscope (manual method).
Nowadays, semi-automatic method can be used (with image acquisition and use of Fiji) or totally automatic with the use of coulter counters that provide quick results.

Trypan blue viability test: manual method

Protocol and tips

A. After treatment with the substance of interest,

if the cells are adherent to the growth vessel, you must detach them by the method you usually use (trypsinization or any other methods you use when you have to subculture the cells)
if cells grow in suspension, centrifuge to pellet

TIP 1 FOR ADHERENT CELLS

Note that during trypsinization most of the non-viable cells will be lost in the PBS during the first wash to remove the serum, prior to trypsin treatment.

For an accurate assessment of the number of non-viable cells, it will therefore be necessary to recover the wash PBS by combining it (in the same eppendorf) with the trypsinized cell suspension and pelleting the cells deriving from washing and trypsinization for the viability test.

Trypsin is a rather harsh treatment for detaching cells and could further damage cells upon exposure to a cytotoxic agent. For this reason it is better to choose the gentlest possible subculture method that may not be trypsin

B. resuspend the pelletted cells in the growth medium so that the cell suspension have a cell concentration between 2×10⁶ and 10⁶ cells / ml

TIP 2

This value of cell concentration is chosen in order to have a good number of cells (between 100 and 50) subsequently in the hemocytometer grid.

It is an indicative value that allows you to have not too many and not too few cells to count, in order to minimize the error having reasonable accuracy and precision.

Furthermore, for the subsequent count it is imperative that the cells are well separated (avoid cell clumping) from each other.

C. Make a 1: 1 dilution of the cell suspension with 0.4% trypan blue solution and smoothly mix.

In practice, after mixing the cell solution carefully and thoroughly (remember that the cells settle very quickly!) take 50 µl of the cell suspension, put them in an eppendorf and add an equal volume (50 µl ) of trypan blue solution. Avoid foaming

TIP 3

The 50 µl volume of cell suspension and trypan is only indicative, you can choose a desired volume. However, the volume must not be too small so that a deep mixing between the solutions is guaranteed, possibly avoiding the formation of foam!

Before use it is better to filter the trypan solution with a syringe equipped with a 0.2 µm filter as precipitates tend to form which could make it more difficult to identify the cells.

This is absolutely imperative when using an automatic coluter counter.

D. leave the samples for 5 min at room temperature.

TIP 4

Trypan blue is toxic for the cells! do not leave cells in trypan blue for more than 15 min. Try to maintain the same incubation time with trypan blue for the different samples.

E. Prepare the hemocytometer and count the cells as indicated here

F. Count cells in at least 4 quadrants, distinguishing blue (dead) from unstained (viable) cells. Take an average of the value obtained

TIP 5

Since you have made a dilution of the initial cell suspension with equal volume of trypan, it will be necessary to multiply the value of cells / ml by 2 to get the total number of cells in the cell suspension.

G. Calculate the% of viable and dead cells

% viable cells = (number of unstained cells / total number of cells)x100

% dead cells = (number of blue cells / total number of cells)x 100

H. Repeat the counting procedure at least two more times by repeating steps D to F

Trypan blue viability test: semi-authomatic method with Fiji

This method is completely analogous, in the cell preparation phase, to the previous one.

However, it does require image capture with a micro camera.

Advantages:

you can save images and results for future documentation
It allows to increase the speed of the counts
It also improves the accuracy of the count performed by using an analysis program after acquisition (a posteriori).

Fiji is a totally free program that can be downloaded here. Fiji is an image processing package with a lot of plugins that facilitate image analysis. Here we will use the Cell count plugin

Protocol

Proceed as above from A to E.

F. load the cell suspension into the hemocytometer, focus and acquire the image (or images relating to the different treatments whose cytotoxicity is to be evaluated).

TIP 1

Since you carry out the cell count a posteriori, you can to prepare a certain number of samples at the same time for rapid acquisition one after the other..

G. Open Fiji

H. Open the first image you captured (File –> Open)

I. Activate the Plugin Cell Counter (Plugins –>Analyze–>Cell Counter). Flag on “Keep original”,and click on Initilize. A duplicate of your image will be created.

J. Select the Type 1 counter and click Rename. A dilaog box will appear where you can rename your counter Type, let’s call it “alive” for example

K. Rename also the Type 2 counter with “dead”

L. Now select the alive counter, go on the original image and click on each of the alive cell that you can see in your image (number 1 on the image). Then select the “dead” counter and do the same with the blue coloured cells. You’ll end up with all the cells selected.

M. Click on “result”. So that you’ll end up with a table showing the number of dead cells and the number of alive cells

N. Calculate the % of viable cells

%viable cells=[Number of alive cells/(number of alive cells/number of dead cells)]x100

Trypan blue viability test for monolayer culture

Trypan blue viability test can be performed directly to asses viability in monolayered culterd cells without the need to detach them from culture vessels.

With this method there is no risk of damaging the cells due to trypsinization.

However, for this method of cell counting of monolayer culture, erythrosin B is preferable to trypan.

I mention the protocol here because you can find trypan easily in a cell culture lab, and, ultimately, it is not so bad especially if combined with image analysis with fiji

PROTOCOL

After treatment with the substance of interest

A. Add a 1:10 dilution (i.e. 100 µl in 1 mL of medium) of the 0.4% trypan blue solution to the well where the cells are growing. Mix by pipetting up and down gently, carefully and avoiding foaming.

B. incubate 5 minutes at room temperature

C. Remove the trypan incubation medium and wash 2 times with PBS

D. visualize the cells with a phase contrast microscope
if possible acquire the images and count the cells with fiji, as explained in the previous protocol

Final considerations and limitations

In addition to trypan blue, other dyes such as erythrosin B, naphthalene black, eosin, Congo red or propidium iodide can be used in the dye exclusion test.

While performing the dye exclusion test, some limitations must be kept in mind

irreversibly damaged cells can take several days to lose the integrity of their membrane, so the damage would not be detected with this type of test
some damaged cells may undergo premature disintegration so they are not detected in the dead cell count.

These factors can cause cell death to be underestimated.

Trypan is toxic, both to cell and to you! It’s is carcinogenic and can cause genetic defects. Be careful when handling

Contents